In this study, we obtained an oral cancer cell line, HSC-3, from the Health Science Research Resources Bank (Osaka, Japan). This cell line originates from a metastatic deposit of a poorly differentiated squamous cell carcinoma (SCC) of the tongue, specifically in a mid-internal jugular lymph node, extracted from a 64-year-old man. The cultivation of HSC-3 cells was performed using Eagle’s minimum essential medium (Sigma–Aldrich, St. Louis, MO, USA), which was supplemented with fetal bovine serum (10%; Thermo Fisher Scientific, Waltham, MA, USA), as well as a solution of penicillin and streptomycin (1000 units/mL; Sigma–Aldrich). Trypsin (0.25%; FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) and ethylenediaminetetraacetic acid (0.02%; DOJINDO LABORATORIES, Kumamoto, Japan) solutions were used to isolate cells for subculture, as previously described [24 (link)].
Ethylenediaminetetraacetic acid
Manufactured by Dojindo Laboratories
Sourced in Japan
Ethylenediaminetetraacetic acid (EDTA) is a chelating agent used in various laboratory applications. It forms stable complexes with metal ions, effectively removing or sequestering them from solutions.
Automatically generated - may contain errors
Lab products found in correlation
Bz 9000, by Keyence (2 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Il 10, by Thermo Fisher Scientific (2 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions)
Trypsin, by Fujifilm (1 mentions)
Hsc 3, by Merck Group (1 mentions)
Eagle s minimum essential medium, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Hsc 3, by Health Science Research Resources Bank (1 mentions)
Citric acid monohydrate, by Kanto Chemical (1 mentions)
Noradrenaline, by Merck Group (1 mentions)
Sodium acetate, by Kanto Chemical (1 mentions)
Isoproterenol, by Merck Group (1 mentions)
Phenylmethylsulfonyl fluoride, by Thermo Fisher Scientific (1 mentions)
Ultrasonic disintegrator, by Sonics (1 mentions)
Timp 2, by RayBiotech (1 mentions)
Igf 1, by Mediagnost (1 mentions)
Qubit protein assay kit, by Thermo Fisher Scientific (1 mentions)
Tgf β1, by R&D Systems (1 mentions)
8 protocols using ethylenediaminetetraacetic acid
1
Oral Cancer Cell Line HSC-3 Cultivation
2
HPLC Analysis of Catecholamines
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(2S,3S)-Desethylreboxetine was purchased from ABX (Radeberg, Germany). Methanol was HPLC grade from Merck. Noradrenaline, 3,4-dihydroxybenzylamine, and isoproterenol were purchased from Merck, citric acid monohydrate and sodium acetate from Kanto Chemical (Tokyo, Japan), ethylenediaminetetraacetic acid from Dojindo (Kumamoto, Japan), and sodium octane sulfonate from FUJIFILM Wako Pure Chemical (Osaka, Japan). All other chemicals were of analytical grade and were used without further pretreatment. Stock solutions of Noradrenaline, 3,4-dihydroxybenzylamine and isoproterenol were prepared separately at a concentration of 1 mg/mL in 0.2 M perchloric acid at -80 °C.
3
Quantification of aortic tissue proteins
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Aortic tissues and 20 μM montelukast-induced macrophages were homogenized using an ultrasonic disintegrator (Sonics & Materials, Inc., Newtown, CT, USA) in protein extraction buffer (CytoBuster; Novagen, Merck KGaA, Darmstadt, Germany) with 20 mM of ethylenediaminetetraacetic acid (Dojindo, Kumamoto, Japan) and 1 mM of phenylmethylsulfonyl fluoride (Thermo Fisher Scientific). Lysate protein concentration was measured using the Qubit® Protein Assay Kit and Qubit® 2.0 fluorometer (Thermo Fisher Scientific). An equal concentration of total protein was applied to each assay kit and detected according to the manufacturer's protocol for each ELISA kit (TIMP-1 and TGF-β1: R&D Systems, Minneapolis, MN, USA; TIMP-2: RayBiotech, Norcross, GA, USA; IGF-1: Mediagnost, Reutlingen, Germany; IL-1β, IL-6, TNF-α, and MCP-1: Bender MedSystems, Vienna, Austria; IL-10: Thermo Fisher, Waltham, MA, USA) [12 (link)]. Seven samples were analyzed per group.
4
Measurement of 8-isoprostane in skin
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The skin tissue specimens were homogenized with 0.1 M phosphate (pH 7.4) containing 1 mM of ethylenediaminetetraacetic acid (Dojindo Laboratories, Kumamoto, Japan) and 50 µg/mL (w/w) of dibutylhydroxytoluene (Wako). The homogenate was centrifuged at 8,000×g for 10 minutes at 4°C, and the total supernatant was used for the assay. The 8-isoprostane concentration of the homogenate was measured using an 8-isoprostane enzyme immunoassay kit (Cayman Chemical Company, MI, USA) according to the manufacturer's instructions. The protein concentration of the supernatant was assessed using the DC protein assay kit (BioRad, Hercules, CA, USA), and the 8-isoprostane level was normalized to the protein level.
5
ZFTA Promoter Luciferase Assay
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The upstream sequence of ZFTA from − 3149 to +2 (corresponding to the second nucleotide of ZFTA’s first codon) was amplified by PCR using a human bacterial artificial chromosome clone (RP11-179P19, Advanced GenoTechs), which lacks the − 154th guanine base, as a template, and cloned into a pGL4.26 (luc2/minP/Hygro) vector (Promega). A series of deletion mutants for this plasmid was generated by PCR using PrimeSTAR MAX or Tks Gflex DNA polymerase (TaKaRa). HeLa cells were transfected with the indicated plasmids listed in Table S1 using HilyMax (Dojindo) and incubated overnight. Luciferase assay was performed using TriStar LB 941 (Berthold) and the following reaction buffers: 100 mM Tris–HCl (pH7.8, Nacalai Tesque), 5 mM MgCl2 (Wako Pure Chemical), 250 µM Coenzyme A trilithium salt (Oriental Yeast), 150 µM adenosine triphosphate (Nacalai Tesque), 150 µg/mL D-Luciferin potassium salt (Cayman Chemical), 0.5 mM dithiothreitol (Nacalai Tesque), 50 µM ethylenediaminetetraacetic acid (pH8.0, Dojindo) and 0.1% Triton X-100 (Sigma-Aldrich).
6
Quantifying Maxillary Bone Necrosis After Extraction
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After examination by μ-CT, the maxillae were demineralised with 20% ethylenediaminetetraacetic acid (Dojindo Laboratories, Kumamoto, Japan) and dehydrated with a graded ethanol series (99% Synthetic Ethanol; Mitsubishi Chemical, Tokyo, Japan) and xylene (Nacalai Tesque, Kyoto, Japan), then embedded into paraffin. Sections (3 μm thick) were cut parallel to the coronal plane and stained with Ladewig’s fibrin stain37 (link),41 (link). Sections were examined by light microscopy (BZ-9000; Keyence, Osaka, Japan). The length of necrotic bone exposed toward the oral cavity, distance between the edges of the epithelial surfaces, area of necrotic bone and necrotic bone ratio were calculated for three sections of the maxillae, namely the midsection of the extraction socket and sections 100 μm mesial and distal to the midsection. The area of necrotic bone was defined as the area of the bone with empty osteocytic lacunae. The necrotic bone ratio was defined as the ratio of empty to occupied osteocytic lacunae within a representative square of 2500 μm2 at the exposed area of the bone42 (link). All measurements were done three times and the average calculated.
7
Histological Analysis of Mouse Knee Joints
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Mouse whole knee joints were harvested at weeks 1 and 4, fixed with 4% paraformaldehyde (Fujifilm Wako Pure Chemical Corp., Osaka, Japan), demineralised with ethylenediaminetetraacetic acid (Dojindo Laboratories, Kumamoto, Japan), and embedded in paraffin to prepare 5-μm-thick sections of frontal plane tissue. Histological sections were visualised under a fluorescence microscope (BZ-9000; Keyence, Osaka, Japan).
8
PDMS Microwell Cell Culture Platform
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Chemicals Dulbecco's modified Eagle's medium (DMEM, low glucose type) was obtained from Nissui Pharmaceutical Co., Ltd. (Tokyo, Japan). Fetal bovine serum (FBS) was obtained from Equitech-Bio, Inc. (Kerrville, TX, U.S.A.). A PDMS prepolymer and the curing agent (Silpot 184) were purchased from Toray-Dow Corning Co. (Tokyo, Japan). PNIPAAm was obtained from Polysciences Inc. (Warrington, PA, U.S.A.). Trypan blue stain solution (0.5%) was obtained from Nacalai Tesque Inc. (Kyoto, Japan). Trypsin was obtained from Becton Dickinson and Co. (Mansfield, MA, U.S.A.). Ethylenediaminetetraacetic acid, disodium salt, dihydrate (EDTA-2Na) was obtained from Dojindo Laboratories (Kumamoto, Japan). All other chemicals were of the highest grade commercially available.
Cells and Cell Culture HepG2 cells were purchased from American Type Culture Collection (Manassas, VA, U.S.A.) and grown in DMEM supplemented with 10% heat-inactivated FBS, 0.15% NaHCO 3 , 100 U/mL potassium penicillin G, and 100 mg/mL streptomycin sulfate at 37°C in humidified air containing 5% CO 2 .
Preparation of PDMS-Based Microwells Four types of micropillar array with different widths, i.e., 360, 450, 560, and 770 µm, were fabricated as previously described. The microwells were named small, medium, large, and extra large according to their size.
Cells and Cell Culture HepG2 cells were purchased from American Type Culture Collection (Manassas, VA, U.S.A.) and grown in DMEM supplemented with 10% heat-inactivated FBS, 0.15% NaHCO 3 , 100 U/mL potassium penicillin G, and 100 mg/mL streptomycin sulfate at 37°C in humidified air containing 5% CO 2 .
Preparation of PDMS-Based Microwells Four types of micropillar array with different widths, i.e., 360, 450, 560, and 770 µm, were fabricated as previously described. The microwells were named small, medium, large, and extra large according to their size.
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